Isolation, identification, and biochemical characterization of five Lacticaseibacillus strains from Oggtt: A traditional fermented and dried buttermilk

Abstract This study investigates the isolation and characterization of the main lactic acid bacteria responsible for fermentation of Oggtt, a dried fermented buttermilk. Five isolates with Gram‐positive staining and negative catalase and oxidase activity were identified using phenotypic and genotypic methods, and their antagonistic, exopolysaccharides and organic acid production, proteolytic activity, and antioxidant capacity were assessed. The isolates are classified as Lacticaseibacillus paracasei Ogt_1, Lacticaseibacillus casei Ogt_2, Lacticaseibacillus paracasei Ogt_3, Lacticaseibacillus paracasei Ogt_4, and Lacticaseibacillus paracasei Ogt_5. All strains possessed high antagonistic activity against Proteus vulgaris, Staphylococcus aureus, and Escherichia coli. All strains produced high levels of lactic acid (11177.3–15404.9 μg/ml), tartaric acid (2197.8–4058.5 μg/ml), and exopolysaccharides(20.86–239.9 mg/L) and possessed high proteolytic and antioxidant activity at variable manners. Overall, this study indicates the isolation of important Lacticaseibacillus strains from Oggtt, which could be used as starter cultures for developing functional foods.


| INTRODUC TI ON
Lactic acid bacteria (LAB) are a crucial group of bacteria that are able to yield lactic acid through hetero-or homofermentative metabolism (Mohamad et al., 2020). Several studies have reported that LAB are able to produce diverse beneficial metabolites including short-chain fatty acids, essential amino acids, digestive enzymes, water-soluble vitamins, and antimicrobial agents (Amin et al., 2020;Masuda et al., 2012). The fermentation and enzymatic functions of LAB are considerable for texture, flavor, inhibition of spoilage microorganisms, and extended shelf-life of fermented food products (Morales et al., 2011).
Various LAB species have isolated from different sources of food such as bovine milk, camel milk, cheese, yogurt, and as well from the gastrointestinal tract of human, bee, and some animals (Kwun et al., 2020). Although the selection of commercial starter LAB for using in food fermentation is a crucial issue, the screening of novel LAB strains from unprecedented sources may be valuable for human health and food industries. The isolation and identification of novel LAB strains aims to reveal the characteristic taxonomy of LAB, obtain promising beneficial and functional probiotic strains particularly from the genus of Lactobacillus, and ensure the production of standard quality of fermented food products (Celik et al., 2021). Lactobacillus species hold a long history of safe use where they are responsible of the fermentation process together with associated starter cultures or they were added as adjunct cultures (Widyastuti et al., 2021). Most of Lactobacillus strains are non-pathogenic residents in animal and human intestine and their existence is crucial for the protection of gut microflora (Jacobsen et al., 1999).
Oggtt is one of the dried fermented food products with a long shelf-life due to its a low pH and water activity that is produced traditionally in several Middle East countries including Saudi Arabia, Jordan, Syria, Iraq, and Egypt. It is characterized by high protein and calcium content, low fat percentage, and pleasant organoleptic properties (Hamad et al., 2016). Oggtt is still produced under no modern or standardized protocol. Although Oggtt, Madeer, and Jameed are sun-dried fermented buttermilk prepared after spontaneously fermented and churning goat, camel, or sheep milk, there is a little difference in processing steps between them (Al-Abdulkarim et al., 2013). In Saudi Arabia, Oggtt is made by boiling the buttermilk with stirring until it thickens, then thick paste is allowed to cool and shaped before let to the sun for drying (Al-Abdulkarim et al., 2013;Hamad et al., 2016). In Jordan, Syria, Iraq, and Egypt, the produced buttermilk is let to drain in cheesecloth instead of boiling before shaping and allowing for solar drying (Alu'datt et al., 2016). In both methods, the fermentation is done spontaneously by wild LAB (Hamad et al., 2016). So, these strains can be isolated and identified to be used as pure culture strains for further milk fermentation.
However, to date studies on the isolation and characterization of lactic acid bacteria (LAB) from Oggtt are scarce (Al-Hindi et al., 2015) and consequently the main strains involved in the fermentation of Oggtt remain anonymous. Therefore, the aim of the present work was to isolate and identify novel LAB from Oggtt that produced indigenously in Saudi Arabia.

| Materials
In Saudi Arabia, there are two types of Oggtt that differed in their color due to variations in manufacturing seasons. The first type is the brown color Oggtt, which is manufactured in summer, while the second type is the creamy color Oggtt which is manufactured in winter. In this study, 40 Oggtt samples (20 brown color Oggtt and 20 creamy color Oggtt) weighing about 200 g each were collected from popular stores in Qassim region and aseptically handled to avoid contamination and transferred in sterilized containers to the laboratory for analysis. All chemicals and microbiological media were of analytical grades and were obtained from Sigma-Aldrich (Egyptian International Center for Import) and Oxoid Limited Company, respectively.

| Biochemical screening
The presumptive Lacticaseibacillus isolates were distinguished using Gram staining, cell morphology, catalase activity, oxidase activity, milk coagulation, and gas production from glucose in MRS broth as previously described by Patil et al. (2010). Under safety conditions, the non-LAB isolates were discarded. Gram-positive isolates exhibited negative catalase and oxidase activity was maintained in MRS broth supplemented with 15% glycerol before being stored at −20°C.

| Molecular characterization of isolated LAB by 16S rRNA gene sequencing
Bacterial identification by polymerase chain reaction (PCR) and DNA Sequencing of 16S rRNA gene was done. The Lacticaseibacillus strains identification was confirmed by 16S rRNA sequence analysis. Initially, chromosomal DNA was extracted from each isolate according to the method mentioned by Ward and Downie (2005). The polymerase chain reaction (PCR) primer used were as follows: 28F 5′AGAGTTTGATCCTGGCTCAG-3′ (positions 8-28 in E. coli numbering) and 1512 R 5′ACGGCTACCTTGTTACGACT-3′ (positions 1512-1493 in E. coli numbering). PCR amplifying protocol was as follows; initial denaturation for 3 min at 95°C for 1 cycle, 40 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min, and a final extension cycle of 72°C for 10 min.
PCR amplified DNA segments were separated by electrophoresis on 0.8% agarose gel and visualized using ethidium bromide.
Product size was determined by comparison with a DNA 1 KB ladder. Products were purified using QIA quick PCR Purification Kit according to the manufacturer's instructions. For verifying the purification, the purified products were run on 0.8% agarose gel before the Sequencing of the PCR fragments which was conducted by Cairo University Research Park (a commercial service provider).
Databases were screened for similarities by using BLAST program (NCBI). A Phylogenetic tree was constructed based on 16S rRNA gene sequences of the five strains using the 'Phylo geny.fr' online tool (Dereeper et al., 2008). The tree showed the relationship between the isolates and other closely related sequences of NCBI GenBank reference taxa.

| Organic acid profile of isolated LAB
The organic acids in the cell-free supernatant (CFS) were analyzed according to the method described by Parlindungan et al. (2021). An amount of 20 μl of the sample was injected into an Agilent 1200 highperformance liquid chromatography (HPLC) system with a Refractive Index Detector and a REFEX 8 μm 8% H Organic Acid Rezex@ column (Phenomenex). The elution fluid was H 2 SO 4 (5 mmol/L) at a flow rate of 0.6 ml/min with the temperature of the column retained at 65°C. MRS broth was also analyzed as a control. Authentic standards of organic acids were run under the same conditions, and the retention time of peaks of the samples was compared with those of organic acid standards and quantified by calculating area under the peaks.

| Exopolysaccharides (EPS) of isolated LAB
EPS were determined following the method described by Welman and Maddox (2003). Briefly, LAB isolates were grown in MRS broth at 37°C for 16-18 h to attain absorbance (A600) of 0.9, and then inoculated at 1% (v/v) in modified MRS broth, in which, glucose (20 g/L) was replaced with sucrose, and fermentation was allowed to proceed for 72 h at 37°C. Then, modified MRS agar was inoculated with 1% (v/v) fresh culture using pour plate technique and the stickiness of colonies grown in modified media agars was determined by the inoculating loop method. EPS were assayed in the supernatant.
Culture supernatant obtained after centrifugation (11,000 g for 10 min) was added to two volumes of cold ethanol, and allowed to stand for 12 h at 4°C until precipitation take place. The precipitate was separated by centrifugation (2500 g, 20 min) and suspended in distilled water, followed by the addition of 2 volumes of cold ethanol.
Samples were centrifuged at 2500 g and the EPS pellets were dried at 60°C and weighed.

| Proteolytic activity of isolated LAB
The Proteolytic activity of the identified Lacticaseibacillus strains was evaluated using the method of Shori et al. (2013). Briefly, 10 g of

| Antioxidant activity of isolated LAB
The antioxidant activity of milk fermented by different identified Lacticaseibacillus isolates was estimated using DPPH radical scavenging activity and metal ions chelating activity according to the methods described by Jemil et al. (2016).

| Antagonistic activity of isolated LAB
The antagonistic activity of the pre-identified Lacticaseibacillus strains against some pathogenic microbes was also evaluated. The pathogenic bacteria used were: Escherichia coli ATCC 25922, Proteus vulgaris ATCC13315, Staphylococcus aureus ATCC25923, and Bacillus subtilis NRRL B-543, as well as 2 fungal strains (Aspergillus fumigatus RCMB 002008 and Candida albicans ATCC25923). The antagonistic activity was evaluated for separate strains using the agar well diffusion. Briefly, 100 μl of the pathogenic bacteria suspension (10 8 CFU/ ml) was spread over the entire agar surface of Mueller Hinton (MH) and a 6-mm diameter well was punched aseptically onto the MH agar. After that, 100 μl of MRS broth containing an overnight cultured LAB isolates was seeded into the well and the plate was incubated at 37°C for 24-48 h. Inhibition zones diameter indicating antagonistic activity of LAB were measured with a ruler held against the back of the Petri plate and the measurements were calculated in millimeter (mm) as follow: The obtained results were taken as mean ± SD and calculated from three independent experiments. As previously described, the inhibition zone was scored as strong inhibition, moderate inhibition, and weak inhibition if the diameter was >6 mm, between 3 and 6 mm and <3 mm, respectively (Pan et al., 2009).

| Isolation and identification of LAB
In this study, 25 isolates of LAB were obtained from Oggtt samples.
Of them, five isolates (Ogt_1, Ogt_2, Ogt_3, Ogt_4, and Ogt_5) with different morphological characteristics were selected for further analysis. Upon incubation of the isolates in MRS Microaerophiles agar media for 24 h, creamy, opaque, and circular colonies were observed for the isolates Ogt_1, Ogt_3, and Ogt_4, whereas Ogt_2 exhibited milky white, rounded, and smooth colony shape and Ogt_5 colonies appeared as grayish-white in color with smooth edges ( None of the five isolates produced carbon dioxide from glucose and these results confirmed the homofermentative characteristics of the isolated strains (Galli et al., 2022). Homofermentative LABs are of high importance for the production of high-quality lactic acid for food and pharmaceutical applications in addition to their probiotic properties (Galli et al., 2022;Moon et al., 2012).
In order to identify the five chosen isolates, partial 16S rRNA sequence analysis was conducted using two universal primers (Weisburg et al., 1991), following phenotypic characterization.
Each of the chosen isolates generated a PCR product at 1500 bp ( Figure 1a). These PCR products were then purified using QIA quick PCR Purification Kit and DNA sequencing of the purified products was done. The GenBank database (NCBI) was then used to search for 16S rRNA sequence similarities and the sequence analysis (BLAST) of all isolates clearly indicated the identity of the organisms. Further evidence of the closeness of the isolates to Lacticaseibacillus was obtained by reconstructing a phylogenetic tree ( Figure 1b). The robustness, reliability, and ease of construction of the Phylogeny employment in the phylogenetic trees made it a valuable tool in tree reconstruction (Dereeper et al., 2008) known for their potential applications in the production of highpurity lactic acid and functional probiotic foods (Galli et al., 2022;Moon et al., 2012). Hence, the identified strains could be of potential application in the production of functional foods.

| Organic acids produced by the isolated strains
Identification of organic acids as one of the important components produced by lactic acid bacteria strains was conducted to understand the mechanisms of action behind antimicrobial and therapeutic characteristics of these bacteria and their ability for producing specific organic acids. Thus, the production of some organic acids such  (Figure 2a). The highest levels of EPS were produced by Lacticaseibacillus paracasei Ogt_1, whereas the lowest values were produced by Lacticaseibacillus paracasei Ogt_5 indicating great differences in the EPS production among the isolated strains (p ≤ .05).
The variation in the EPS among the isolated strains is likely due to the differences in the genetic makeup, EPS production pathways, and EPS types. Interestingly, the values of EPS in the isolated strains are within the range 10-400 mg/L produced by LAB under nonoptimized conditions, which could be increased by twofold under optimal growth conditions (Korcz & Varga, 2021). EPS produced by LAB can affect the stability and sensory quality of fermented milk products as they act as texturizers and stabilizers, increasing the viscosity and mouthfeel of products (Galli et al., 2022;Korcz & Varga, 2021). In addition, EPS have high bioactive properties and possess antioxidants, antimicrobial, antiviral, anticoagulant, immunomodulating, and cholesterol-lowering effects (Zhou et al., 2019).
Thus, the findings of this study indicate the potential application of the isolated strains in the development of functional foods and pharmaceutical products.

| Proteolytic activity of the identified strains
The proteolytic activity of identified Lacticaseibacillus strains cultivated in sterilized skim milk was determined using the OPA method and the results are illustrated in Figure 2b. The proteolytic activity altered among the different Lacticaseibacillus strains.

| Antagonistic activity of identified Lactobacillus strains
The identified Lacticaseibacillus strains were further evaluated for their antagonistic activity against the following pathogenic micro- of these pathogenic strains is likely due to the production of metabolites such organic acids and bacteriocins by the isolated strains.
No antifungal activity was detected against all examined fungi used in this study. In agreement with our findings, previous studies indicated that L. casei and L. paracasei possessed antagonistic activity against various pathogenic bacterial strains (Belguesmia et al., 2020;da Costa et al., 2018;Mechai et al., 2020). The antagonistic activity of these strains was attributed to the production of organic acids (da Costa et al., 2018) and bacteriocins (Belguesmia et al., 2020;Mechai et al., 2020). The antagonistic activity of the isolated strains could indicate the potential applications of these strains as biopreservation tools in fresh food products.

| Principal component analysis (PCA) and hierarchical clustering analysis (HCA)
To deeply assess the overall interrelationship between the isolated strains based on their biochemical, antimicrobial, and antioxidant properties, PCA and HCA were performed the results were presented in Figure 3. In the HJ-biplot ( Figure 3a) the similarity between the isolated strains is indicated by the distance between them, in which, short distance indicates similarity, whereas, long distance indicates dissimilarity (Yan & Fregeau-Reid, 2008 strains exhibited different phenotypic characteristics and application potentials.
Lacticaseibacillus paracasei Ogt_3 outscore all other isolates due to its greater antioxidant, and proteolytic activities and production of good amounts of EPS, and butyric, malic, and acetic acids.

| CON CLUS IONS
In conclusion, no doubt that lactic acid bacteria play a crucial role in food industry and human nutrition. Therefore, the isolation and identification of new LAB strains from novel sources are considered significant to be suitable in manufacturing of fermented food products as adjunct starter cultures. The present study provides proof for opportunities to avail Oggtt product, dried fermented milk, as comparatively unexplored sources for the isolation and identification of novel LAB strains for their possibility to utilize as adjunct cultures. We successfully isolated, identified, and characterized five Lacticaseibacillus strains, and illustrated their antagonistic, exopolysaccharides production, proteolytic and antioxidant abilities. According to 16S rRNA gene sequences, potential LAB was identified as Lacticaseibacillus paracasei Ogt_1, Lacticaseibacillus  Ogt_3 possess good proteolytic and antioxidant activities. The present work proves that Oggtt product is a rich source of LAB that might have a range of various benefits and applications in fermented food industry. Also, this study provides vigorous support for further in vitro and in vivo research to investigate the beneficial effects of identified strains and their utilization in fermented food products.

ACK N OWLED G M ENTS
The authors are thankful to the Faculty of Agriculture, Cairo University, Giza, Egypt for supporting analytical samples at Cairo University Research Park (CURP) and the regional center for mycology and biotechnology. The authors greatly appreciate the suggestions of Dr Samia El-Dieb, Professor of dairy science, Faculty of Agriculture, Cairo University and her valuable effort done in order to support the current research team.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.

E TH I C S S TATEM ENT
These data were generated from a fermented food, and therefore no ethics approval was needed.